Genotype/Phenotype Correlation in Autosomal Recessive Lamellar
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View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Am. J. Hum. Genet. 62:1052–1061, 1998 Genotype/Phenotype Correlation in Autosomal Recessive Lamellar Ichthyosis Hans Christian Hennies,1 Wolfgang Ku¨ster,2 Victor Wiebe,1 Alice Krebsova´,1,3 and Andre´ Reis1 1Institute of Human Genetics, Charite´, Humboldt University, Berlin; 2TOMESA Clinic for Allergy, Skin and Joint Diseases, and Rheumatism, Bad Salzschlirf, Germany; and 3Department of Medical Genetics II, University Hospital Motol, Charles University, Prague Summary Introduction Autosomal recessive lamellar ichthyosis is a severe con- Lamellar ichthyosis (LI) is a heterogeneous group of se- genital disorder of keratinization, characterized by var- vere hereditary disorders of keratinization. Marked dif- iable erythema of the whole body surface and by dif- ferences in the clinical picture and course of LI are ob- ferent scaling patterns. Recently, mutations have been served (Traupe 1989). In most cases, LI is inherited identified in patients with lamellar ichthyosis in the autosomal recessively; a rare form, however, of auto- TGM1 gene coding for keratinocyte transglutaminase, somal dominantly inherited LI has been described as well and a second locus has been mapped to chromosome 2. (Traupe et al. 1984). In the autosomal recessive type of We have now analyzed the genotype/phenotype corre- LI (McKusick 1994; MIM 242100), newborns often lation in a total of 14 families with lamellar ichthyosis. present a pergament-like collodion membrane. After los- Linkage analyses using microsatellites in the region of ing this encasement, patients show a characteristic gen- the TGM1 gene confirmed genetic heterogeneity. In pa- eralized scaling on the entire body, including scalp and tients not linked to the TGM1 gene, the second region flexural surfaces, and a variable erythema. Various clas- identified on chromosome 2 and a further candidate re- sifications of LI forms have been proposed, according gion on chromosome 20 were excluded, confirming as to clinical, histological, and ultrastructural parameters well the existence of at least three loci for lamellar ich- (reviewed in Traupe 1989). Depending on the finding of thyosis. Sequence analyses of the TGM1 gene in families erythema, two major types of recessive LI were clinically compatible with linkage to this locus revealed seven dif- discriminated (Hazell and Marks 1985; Williams and ferent missense mutations, five of these unpublished so Elias 1986). The erythematous form of LI, also referred far, and one splice mutation. No genotype/phenotype to as nonbullous congenital ichthyosiform erythro- correlation for mutations in the TGM1 gene was found derma, is characterized by a strong redness of the entire in this group of patients, which included two unrelated body surface. Patients often also show an ectropion. In patients homozygous for the same mutation. Similarly, most cases, this form of LI is associated with a fine, no clear difference in the clinical picture was seen be- whitish scaling of the skin and marked palmoplantar tween patients with TGM1 mutations and those un- hyperkeratosis and hyperlinearity (Traupe 1989). In con- linked to the TGM1 locus. Comparison of genetic and trast, a nonerythematous form was described that often clinical classifications for patients with lamellar ichthy- exhibits large dark scales and a rough, barklike pattern osis shows no consistency and thus indicates that clinical of the skin (Williams and Elias 1986). A divergent clas- criteria currently in use cannot discriminate between the molecularly different forms of the disease. sification of patients with LI is obtained on the basis of histological and ultrastructural investigations. Five types of LI can be distinguished by assessment of ultrastruc- tural depositions in the stratum corneum and of the pat- terns of membranes and lamellar structures (Anton- Lamprecht 1992). The horny cells of the stratum corneum are charac- terized by a stable cell envelope formed of several com- Received August 5, 1997; accepted for publication February 27, 1998; electronically published April 17, 1998. ponents that are covalently bound to each other. These Address for correspondence and reprints: Dr. Andre´ Reis, Institut components include a number of proteins, such as in- fu¨ r Humangenetik, Charite´ Campus Virchow-Klinikum, Humboldt- volucrin, loricrin, and the small proline-rich proteins, Universita¨t zu Berlin, Augustenburger Platz 1, D-13353 Berlin, Ger- along with lipids on the outer surface of the cell enve- many. E-mail: [email protected] ᭧ 1998 by The American Society of Human Genetics. All rights reserved. lope. Keratinocyte transglutaminase (TGK), which cat- 0002-9297/98/6205-0009$02.00 alyzes the formation of covalent e-(g-glutamyl)lysine 1052 Hennies et al.: Genotype/Phenotype Correlation in LI 1053 bonds, is involved in the cross-linking of structural pro- (Compton et al. 1992) and with two microsatellites at teins in the upper granular layer. TGK is encoded by the anonymous loci, D14S64 and D14S264 (Dib et al. transglutaminase 1 gene (TGM1) on chromosome 1996), closely linked to TGM1. On chromosome 20, 14q11 (Kim et al. 1992; Phillips et al. 1992; Yamanishi microsatellite markers at D20S101, D20S107, and et al. 1992). Moreover, epidermal transglutaminase, D20S119 were analyzed. Genotyped markers on chro- which is encoded by the transglutaminase 3 gene mosome 2 are at D2S116, D2S325, D2S157, and (TGM3), is present in suprabasal keratinocytes. TGM3 D2S143 (Dib et al. 1996). Analyses were performed by has been localized to chromosome 20q11-q12 (Gentile use of an automated DNA sequencer (ALF, Pharmacia, et al. 1994; Wang et al. 1994). or 373, Perkin Elmer). Microsatellites were amplified in In patients with autosomal recessive LI, a lack of TGK 15-ml PCR mixtures containing 30 ng DNA, 0.33 mM expression has been shown (Hohl et al. 1993; Huber et of each primer, one of which was end-labeled with fluo- al. 1995a; Lavrijsen and Maruyama 1995), and linkage rescent dye, 0.1 mM of each dNTP, and 0.5 U Taq DNA of autosomal recessive LI to TGM1 has been described polymerase (Perkin Elmer) in 50 mM KCl, 10 mM Tris/ (Russell et al. 1994). Subsequently, mutations have been HCl (pH 8.0), and 1.5 mM MgCl2. Reactions consisted identified in the TGM1 gene in patients with LI (Huber of 27 cycles of 15 s at 94ЊC, 15 s at annealing temper- et al. 1995a; Parmentier et al. 1995; Russell et al. 1995). ature, and 30 s at 72ЊC. PCR products were separated In a number of families with autosomal recessive LI, on 6.6% polyacrylamide sequencing gels and were an- however, mutations in the TGM1 gene could be ruled alyzed by use of the Fragment Manager (Pharmacia) or out either by showing normal TGK activity (Huber et the Genotyper software (Perkin Elmer). al. 1995b; Lavrijsen and Maruyama 1995) or by ex- cluding linkage to TGM1 (Parmentier et al. 1995; Bale Linkage Analysis et al. 1996). Furthermore, a second locus for LI (ICR2B) Linkage analyses were performed with the computer has recently been mapped, in three consanguineous fam- programs in the LINKAGE package, version 5.1 (La- ilies from Morocco, to a 6.6-cM interval between the throp et al. 1984). Independent segregation of the phe- markers at D2S325 and D2S137 on chromosome 2q33- notype in question and a locus analyzed is considered q35 (Parmentier et al. 1996). significant for LOD scoresZ ! Ϫ2 . Autosomal recessive Here we present the investigation of a total of 14 inheritance with complete penetrance was assumed in families with clinically different forms of autosomal re- all LI families investigated. cessive LI. Our findings confirm that LI is genetically heterogeneous but do not show consistency of the mo- Mutation Analysis lecular results with clinical classifications. Mutations in the TGM1 gene were identified in a number of LI pa- The translated exons 2–15 of the TGM1 gene were tients, but no genotype/phenotype correlation could be amplified by PCR with intronic primers designed ac- derived. cording to published TGM1 sequences (Kim et al. 1992; Phillips et al. 1992). DNA was amplified by PCR as Material and Methods described above. For SSCP analyses, ∼10% of the vol- ume of the PCR sample was denatured and separated Patients with LI on 8% polyacrylamide gels according to the procedure described by Orita et al. (1989). Electrophoresis was A total of 14 families with autosomal recessive LI were performed at 40 W and 4ЊCor15ЊC for 1.5–6 h. PCR analyzed clinically and molecularly. Thirteen of these products were sequenced directly either by use of a cycle- originate from Germany, and one is of Moroccan ex- sequencing protocol (Amersham) or by solid-phase se- traction. In two families, only one patient each and the quencing with magnetic beads (Dynal) and T7 DNA patient’s parents were available. All others are two- or polymerase (Pharmacia). Sequencing products were an- three-generation families with one or two affected in- alyzed on an automated DNA sequencer (Pharmacia). dividuals. The family from Morocco is consanguineous, A detailed description of the transglutaminase activity an unresolved relationship is remembered in one of the assay will be given elsewhere (M. Huber and D. Hohl, German families, and the parents of all other patients personal communication). In brief, frozen skin sections are unrelated. were incubated in 100 mM Tris/Cl pH 8, 1% BSA for 30 min, to block nonspecific binding, and then in 100 Microsatellite Analysis mM Tris/Cl pH 8, 5 mM CaCl2, and 12 mM monodan- DNA was prepared according to standard methods, sylcadaverine for 1 h, to detect transglutaminase activity. from blood samples drawn from consenting individuals. For a negative control in normal human skin, EDTA Linkage analysis on chromosome 14 was performed was added to a final concentration of 20 mM. After the with the microsatellite located within the TGM1 gene transglutaminase reaction was stopped in PBS/10 mM 1054 Am.